U.S. patent number 8,338,374 [Application Number 10/583,923] was granted by the patent office on 2012-12-25 for stable growth hormone liquid formulation.
This patent grant is currently assigned to Pharmacia Corporation. Invention is credited to Advait Badkar, Sandeep Nema, Manpreet S Wadhwa.
United States Patent |
8,338,374 |
Wadhwa , et al. |
December 25, 2012 |
**Please see images for:
( Certificate of Correction ) ** |
Stable growth hormone liquid formulation
Abstract
The present invention is directed to stable liquid growth
hormone formulations that remain stable after physical agitation,
and after exposure to one or more freeze-thaw events. Formulations
of the present invention can be stored long term at a variety of
temperatures, even frozen. In the present invention, a combination
of buffer and stabilizing agents, including a non-ionic surfactant
(e.g., polysorbate 20), a polymer stabilizer (e.g., polyethylene
glycol), and other optional stabilizers combine to provide
unexpected stability to aqueous formulations of a growth hormone
(e.g., human growth hormone).
Inventors: |
Wadhwa; Manpreet S (Thousand
Oaks, CA), Nema; Sandeep (St. Louis, MO), Badkar;
Advait (Wildwood, MO) |
Assignee: |
Pharmacia Corporation (St.
Louis, MO)
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Family
ID: |
34738712 |
Appl.
No.: |
10/583,923 |
Filed: |
December 13, 2004 |
PCT
Filed: |
December 13, 2004 |
PCT No.: |
PCT/IB2004/004159 |
371(c)(1),(2),(4) Date: |
May 14, 2007 |
PCT
Pub. No.: |
WO2005/063298 |
PCT
Pub. Date: |
July 14, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20080125356 A1 |
May 29, 2008 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60531843 |
Dec 23, 2003 |
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Current U.S.
Class: |
514/11.4;
530/351; 424/198.1; 424/497 |
Current CPC
Class: |
A61P
5/06 (20180101); A61P 19/10 (20180101); A61K
47/12 (20130101); A61K 9/0019 (20130101); A61K
47/10 (20130101); A61P 43/00 (20180101); A61K
38/27 (20130101); A61K 47/26 (20130101) |
Current International
Class: |
A61K
38/27 (20060101); A61K 38/18 (20060101); A61K
47/42 (20060101); A61K 38/17 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO9403198 |
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Feb 1994 |
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WO |
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WO9714430 |
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Apr 1997 |
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WO |
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WO 97/29767 |
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Aug 1997 |
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WO |
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WO9729767 |
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Aug 1997 |
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WO |
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WO9739768 |
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Oct 1997 |
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WO |
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WO9915193 |
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Apr 1999 |
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WO |
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WO 0103741 |
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Jan 2001 |
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WO |
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WO0217957 |
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Mar 2002 |
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WO |
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Other References
Feng et al, Biotechnology Techniques, Apr. 1998, vol. 12, No. 4,
pp. 289-293. cited by examiner .
Bio-Rad laboratories, product sheet, 2000, pp. 1-4. cited by
examiner .
Cleland et al, Pharmaceutical research, 1996, vol. 13, No. 10, pp.
1464-1475. cited by examiner .
Serajuddin et al, Journal of Pharmaceutical Sciences, 1990, vol.
79, No. 5, pp. 463-464. cited by examiner .
Jin Yin et al., Pharmaceutical Research, vol. 21, No. 12., Dec.
2004, pp. 2377-2383. cited by other .
Kozlov et al., Macromolecules, vol. 33, pp. 3305-3313, 2000. cited
by other .
Bam et al., Journal of Pharmaceutical Sciences, 1991, vol. 87, No.
12, p. 1554- 1559. cited by other.
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Primary Examiner: Bunner; Bridget E
Assistant Examiner: Hamud; Fozia
Attorney, Agent or Firm: Wichtowski; John A.
Parent Case Text
The present application claims priority under Title 35, United
States Code, .sctn.119 to U.S. Provisional application Ser. No.
60/531,843 filed Dec. 23, 2003, which is incorporated by reference
in its entirety as if written herein.
Claims
What is claimed is:
1. A formulation consisting of a therapeutically effective amount
of a human growth hormone in an aqueous solution, a buffer that
maintains the pH of the formulation at a pH of 5 to 7, a non-ionic
polysorbate surfactant, a polymer stabilizer wherein said polymer
stabilizer is a polyethylene glycol which has a molecular weight in
the range of about 3000 to about 20,000, methionine, and one or
more optional excipients selected from the group consisting of a
divalent cation present in a magnesium salt selected from the group
consisting of magnesium hydroxide, magnesium chloride, magnesium
sulfate, magnesium citrate, and magnesium edentate; a tonicity
agent; and a preservative, wherein the formulation remains stable
after at least one freezing and subsequent thawing event.
2. The formulation of claim 1, wherein the human growth hormone is
a recombinant form of human growth hormone.
3. The formulation of claim 2, wherein the recombinant form of
human growth hormone is present in the formulation at a
concentration of 0.1 mg/ml to 20 mg/ml.
4. The formulation of claim 1, wherein the buffer is selected from
the group consisting of sodium citrate, sodium edentate, sodium
succinate, and histidine hydrochloride.
5. The formulation of claim 1, wherein the non-ionic polysorbate
surfactant is present at a concentration of about 0.02% to about
10%.
6. The formulation of claim 1, wherein the non-ionic polysorbate
surfactant is selected from the group consisting of polysorbate 20
and polysorbate 80.
7. The formulation of claim 1, wherein the polyethylene glycol is
present at a concentration of about 0.25% or about 1%.
8. The formulation of claim 1, wherein the tonicity agent is
sorbitol.
9. The formulation of claim 1, wherein the preservative is selected
from the group consisting of phenol and benzyl alcohol.
10. A formulation consisting of about 0.1 mg/ml to about 20 mg/ml
of a recombinant form of human growth hormone in an aqueous
solution, a citrate or edentate buffer that maintains the
formulation at a pH of about 5 to about 7, about 0.04% to about 5%
(w/w) of a polysorbate surfactant, about 0.25% or about 1% (w/v) of
polyethylene glycol, methionine, and one or more optional
excipients selected from the group consisting of a sufficient
concentration of sorbitol for the formulation to be approximately
isotonic, magnesium chloride or magnesium hydroxide, and a
preservative, wherein the formulation remains stable after at least
one freeze thaw event.
11. The formulation of claim 10, wherein the preservative is phenol
or benzyl alcohol.
12. The formulation of claim 10, wherein at least about 90% of the
recombinant form of human growth hormone remains in solution after
exposure of the formulation to three or more freeze-thaw
events.
13. The formulation of claim 10 where the formulation is stable at
about 2.degree. C. to about 8.degree. C. for at least 52 weeks.
14. The formulation of claim 13 wherein after 52 weeks at about
2.degree. C. to about 8.degree. C. at least one of (i) total
aggregate as measured by size exclusion HPLC is less than about
0.5%, (ii) total deamidation as measured by anion exchange HPLC is
less than about 7%, or (iii) the recombinant form of human growth
hormone recovery as measured by reverse phase HPLC is greater than
or equal to 85%.
Description
FIELD OF THE INVENTION
The present invention relates to stable liquid formulations of
growth hormones, such as human growth hormone, particularly, to
such formulations that remain stable after long term storage, and
also remain stable after being subjected to physical stress such as
agitation, freezing, and thawing.
BACKGROUND OF THE INVENTION
Native human growth hormone is a single polypeptide chain protein
consisting of 191 amino acids, internally cross-linked by two
disulphide bridges. The growth hormones of other animal species are
closely homologous to native human growth hormone, and have similar
biological activity in terms of being effective in the treatment of
diseases related to growth hormone deficiencies in humans, such as
hypopituitary dwarfism and osteoporosis. Recombinant forms of human
growth hormone have also been produced with the same or
substantially similar amino acid sequence as native human growth
hormone and identical biological activity to the native hormone.
Except as otherwise noted below, all native and recombinant forms
of human growth hormone are collectively referred to as "hGH."
Because of the structural similarities between hGH and the growth
hormones of other species, one would expect formulations that are
effective in stabilizing hGH to also be effective in stabilizing
the growth hormones of other species.
hGH is primarily sold in lyophilized form today. See, for example,
GENOTROPIN.RTM. Lyophilized Powder (Pharmacia & Upjohn Company,
now owned by Pfizer Inc.), HUMATROPE.RTM. (Eli Lilly),
NORDITROPIN.RTM. for Injection (Novo Nordisk), SAIZEN.RTM. for
Injection (Serono), and NUTROPIN.RTM. (Genentech). Lyophilized
formulations have the advantage of providing protein stability for
long periods of time. However, a lyophilized formulation must be
used shortly after reconstitution, as aggregation and deamidation
tend to begin shortly after reconstitution with an aqueous diluent.
This generally means that it is left up to the consumer of a
lyophilized formulation to reconstitute the product shortly before
use. If reconstitution is not done properly, or if the
reconstituted formulation is stored for too long before use, the
consumer may get an improper dosage of the growth hormone or a
dosage containing unsuitable levels of hGH degradation products. In
addition, manufacture of lyophilized formulations involves
substantially greater cost and time relative to manufacture of
liquid formulations.
Due to the above disadvantages with lyophilized hGH formulations,
various liquid formulations of hGH have been developed over the
years, each with varying degrees of stability under various storage
and handling conditions. Two commercial liquid formulations of hGH
are sold under the brand names NUTROPIN AQ.RTM. (Genentech, Inc.)
and NORDITROPIN.RTM. (Novo Nordisk). The composition of these and
other hGH liquid formulations have been disclosed in issued patents
and published patent applications, summarized below. Each of the
following references states that the liquid formulations of hGH
formulations described therein are stable at refrigeration
temperatures, at about 2.degree. C. to about 8.degree. C., except
where noted otherwise below. However, none claim to disclose
formulations that can withstand exposure to freezing and thawing,
conditions to which products can be exposed in transit.
Furthermore, the hGH in some of the previously disclosed
formulations degrades or undergoes aggregation when subjected to
physical agitation, for example, during shipment. These types of
instabilities not only cause wastage of expensive product, but can
also cause safety issues if the degraded product is inadvertently
administered to a patient.
U.S. Pat. No. 5,567,677 (invented by Castensson et al.; assigned to
PHARMACIA AB) discloses an aqueous formulation consisting of growth
hormone and citrate buffer in an amount of 2-50 mM at a pH of about
5.0 to 7.0. The '677 patent also teaches that mannitol and glycine
can be suitably included in the formulation disclosed therein.
U.S. Pat. Nos. 5,763,394 and 5,981,485 (invented by O'Connor et
al.; assigned to GENENTECH, INC.) disclose an aqueous human growth
hormone formulation containing hGH, a buffer providing pH 5.5 to pH
7 (e.g., sodium citrate), 0.1% to 1% w/v non-ionic surfactant
(e.g., polysorbate 20) and, 50 to 200 mM of a neutral salt (e.g.
sodium chloride), and a preservative (e.g. phenol), wherein said
formulation is free of glycine and mannitol.
U.S. Pat. No. 6,022,858 (invented by Sorensen et al., assigned to
NOVO NORDISK A/S), discloses a buffered aqueous solution containing
a human growth hormone pretreated with zinc salt, and optionally
containing lysine or calcium ion.
U.S. Pat. No. 5,849,704 (invented by Sorensen et al., assigned to
NOVO NORDISK A/S), discloses a buffered aqueous solution containing
a growth hormone buffered with histidine or histidine
derivative.
U.S. Pat. No. 5,977,069, U.S. Pat. No. 5,631,225, and U.S. Pat. No.
5,547,696 (invented by Sorensen et al., assigned to NOVO NORDISK
A/S), disclose buffered aqueous solutions containing a human growth
hormone with stabilizing amounts of the amino acids asparagine,
isoleucine, or valine, respectively.
U.S. Pat. No. 5,705,482 and U.S. Pat. No. 5,552,385 (invented by
Christensen et al., assigned to NOVO NORDISK A/S), disclose
buffered aqueous solutions containing a human growth hormone with
stabilizing amounts of the peptides Leu-His-Leu and Lys-Gly-Asp-Ser
respectively.
WO 01/03741 A1 (for an invention by Siebold et al.; assigned to
GRANDIS BIOTECH GMBH) discloses a "storage stable liquid growth
hormone formulation consisting essentially of growth hormone in
isotonic phosphate buffered solution" and also claims formulations
with phosphate buffer and a non-ionic surfactant present at a
concentration of 0.2% or less. In the Examples section of the
publication, the only non-ionic surfactant used is Pluronic F-68 at
a concentration of 0.2% (w/v) in each of the formulations where it
was included.
WO 02/067989 A1 (for an invention by Seibold et al.; assigned to
GRANDIS BIOTECH GMBH) is directed to "an aqueous growth hormone
formulation comprising growth hormone and (a) citrate buffer of
about pH 5.6 or more, or (b) a buffer other than citrate of about
pH 6.0 or more, and substantially free of crystallization on
storage." The only suitable temperatures for storage of the
disclosed formulations are refrigeration temperature (4.degree. C.
to 8.degree. C.) and above, or in a temperature range of 8.degree.
to 25.degree. C.
U.S. Application Publication No. 2002/0077461 (for an invention by
Bjorn et al.; assigned to NOVO NORDISK OF NORTH AMERICA INC.)
discloses pharmaceutical formulations comprising growth hormone
(e.g., hGH), an amino acid selected from the group consisting of
asparagine, isoleucine, valine, leucine, histidine, a derivative of
histidine, or a peptide comprising at least one basic amino acid
residue and at least one acidic amino acid residue, and a non-ionic
detergent (e.g., a polysorbate or a polyaxamer). The application
also discloses such formulations with a buffer, (e.g. histidine,
citrate, tartrate, or phosphate) for pH 6 to pH 8, a tonicity agent
(e.g., mannitol). The only stability studies disclosed in this
application were carried out at refrigerated temperatures or
above.
WO 01/24814 A1 (for an invention by Chen et al.; assigned to CHIRON
CORPORATION) discloses the use of an amino acid base sufficient to
decrease aggregate formation during storage to stabilize aqueous
polypeptide formulations, where the amino acid base comprises at
least one amino acid selected from the group consisting of
arginine, lysine, aspartic acid, and glutamic acid. This published
application also discloses the inclusion of additional stabilizers
in such formulations, including antioxidants, such as methionine,
and non-ionic surfactants. Interleukin-2 is the only polypeptide
whose stabilization using such a formulation is illustrated
therein.
The commercial formulations of liquid hGH presently available on
the market include phenol as a preservative. See, for example,
NUTROPIN AQ.RTM. (a liquid formulation of recombinant hGH sold by
Genentech Inc.), and NORDITROPIN.RTM. (a liquid formulation of
recombinant hGH sold by Novo Nordisk). However, phenol is known to
promote the aggregation of hGH, especially upon freezing and
thawing (see Maa, Yuh-Fun, et al., Internat J Pharm 140: 155-168
(1996)).
Even with all the advancements that have been made to date in the
development of formulations that stabilize growth hormones in
particular and polypeptides in general, the stability of hGH in
liquid formulations remains a problem. Stability is particularly
problematic in liquid hGH formulations exposed to freezing and
subsequent thawing, especially when a phenolic excipient (e.g.
phenol preservative) is present. Even a single freeze-thaw can
render known liquid hGH formulations, such as the commercial
formulations cited above, unsuitable for human use, due to protein
aggregation and precipitate formation.
There is a need for a liquid hGH formulation that remains stable
under freeze-thaw conditions, as well as under other conditions of
physical stress, such as physical agitation, provided the
formulation also remains stable after long term storage, under
suitable storage conditions. Such a formulation could be stored not
only in a refrigerator, as are the current commercial hGH
formulations, it could also be stored in a freezer.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to a stable liquid formulation
comprising a therapeutic amount of growth hormone in an aqueous
solution, a buffer, a non-ionic surfactant, and a polymer
stabilizer, wherein the formulation remain stable after exposure to
at least one freeze-thaw event. The liquid growth hormone
formulation of the present invention can be frozen and thawed at
least once without visible precipitates being formed in the
formulation, and without significant loss of growth hormone from
solution.
One embodiment of the invention is directed to a formulation
comprising a therapeutically effective amount of growth hormone in
an aqueous solution, a buffer that maintains the pH of the
formulation at a pH of about 5 to about 7, a non-ionic surfactant,
and a polymer stabilizer, wherein the formulation remains stable
after at least one time freezing and subsequent thawing.
Another embodiment is directed to a formulation comprising, about
0.1 mg/ml to about 20 mg/ml of a recombinant form of human growth
hormone in an aqueous solution, a citrate or edetate buffer that
maintains the formulation at a pH of about 5 to about 7, about
0.04% to about 5% (w/w) of a polysorbate surfactant, and about
0.001% to about 20% (w/v) of polyethylene glycol, wherein the
formulation remains stable after at least one freeze thaw event. In
a specific embodiment the formulation remains stable after at least
three freeze thaw events. In a specific embodiment the formulation
remains stable after at least six freeze thaw events.
The liquid formulations of the present invention are stable in the
presence or absence of phenolic preservatives, such as phenol, even
after exposure to multiple freeze-thaw events. This result is
surprising, in view of what is presently known regarding the effect
of phenolic compounds on the aggregation of growth hormone. (See,
e.g., Maa, Yuh-Fun, et al., supra).
The formulations of the present invention are also surprisingly
stable under conditions of physical handling and agitation, such as
the agitation that formulations are exposed to in the process of
being shipped from one part of a country to another, or from one
part of the World to another.
The formulations of the present invention are, furthermore,
surprisingly resistant to degradation during recommended conditions
of long term storage, such as storage under refrigeration from 2 to
8.degree. C. In a specific embodiment the formulation remains
stable for at least 52 weeks of storage at 2 to 8.degree. C. The
present formulations are even resistant to degradation after
storage at temperatures at or below freezing.
As used herein, the terms "human growth hormone" and "hGH" refer to
human growth hormone produced by methods including extraction and
purification from natural human tissue sources, and from
recombinant culture systems transformed with deoxyribonucleic acid
encoding for human growth hormone. The sequence and characteristics
of hGH are set forth, for example, in Hormone Drugs, Gueriguian et
al., U.S.P. Convention, Rockville Md. (1982). The same terms, as
used herein, also refer to agonist analogues of hGH, which contain
substitution, deletion, and/or insertion of amino acids. The same
terms, as used herein, also refer to agonist analogues of hGH
having at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or
99% identity to native 191 amino acid form of hGH. Two species of
hGH of particular note include the 191 amino acid native species
(somatotropin) and the 192 amino acid N-terminal methionine (met)
species (somatrem) commonly obtained through recombinant means.
As used herein, the term "therapeutically effective amount" of hGH
refers to that amount that provides a therapeutic effect in an
administration regimen.
As used herein, the term "freeze-thaw event" refers to exposure of
a liquid solution or other formulation to a temperature less than
its freezing point, typically in a freezer at minus 20.degree. C.
or minus 70.degree. C. until the solution is frozen, followed by
thawing at a temperature greater than its freezing point, typically
at 2 to 8.degree. C. in a refrigerator, or at ambient room
temperature. Samples frozen and thawed two or more times according
to this procedure are said to have undergone multiple freeze-thaw
events.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a bar graph showing the percent of hGH recovered,
measured by size-exclusion HPLC analysis, from commercially
available liquid hGH formulations before and after being exposed to
six freeze-thaw events, as described in Example 1.
FIG. 2 is a bar graph showing the percent of hGH recovered,
measured by size-exclusion HPLC analysis, from liquid hGH
formulations of the present invention after exposure to
freeze-thaws, and after exposure to shipment agitation, as
described in Example 2.
FIG. 3 is a bar graph of the total percent of hGH variants formed,
measured with anion-exchange HPLC, from hGH liquid formulations of
the present invention, in comparison to a previously known hGH
liquid formulation, after six months storage at 5.degree. C., and
after six weeks storage at 25.degree. C., as described in Example
3.
FIG. 4 is a bar graph of the total percent of hGH variants,
measured with anion-exchange HPLC, from hGH liquid formulations of
the present invention, prepared with various buffers, after 18
weeks storage at 5.degree. C., as described in Example 4.
FIG. 5 is a bar graph of the total percent recovery of hGH,
measured using protein absorbance analysis, from hGH liquid
formulations prepared with various tonicity agents, after exposure
to forced physical agitation, as described in Example 5.
FIG. 6 is a bar graph of the total percent recovery of hGH,
measured using protein absorbance analysis, from formulations
prepared with various concentrations of a non-ionic polysorbate
surfactant, after exposure to forced physical agitation, as
described in Example 6.
FIG. 7 is a bar graph of the total percent of hGH variants formed,
measured using reverse phase HPLC, from hGH liquid formulations of
the present invention, prepared with and without methionine, as
described in Example 6, after 31 weeks of storage at 5.degree.
C.
FIG. 8 is a bar graph of the total percent of hGH variants,
measured using reverse phase HPLC, from hGH liquid formulations of
the present invention, prepared with and without magnesium
reagents, after about 2 months of storage at 5.degree. C., as
described in Example 8.
FIG. 9 is a plot of cumulative weight gain observed over time in
hypophectomized rats, after being administered daily injections of
hGH liquid formulations indicated therein, compared with rats
injected with a placebo control solution containing no hGH ("PBS").
--.quadrature.--represents Formulation 6 of Example 3.
--.DELTA.--represents Formulation 5 of Example 3.
--.diamond.--represents Formulation 8 of Example 3.
--.largecircle.--represents PBS control.
FIG. 10 is a bar graph of the total percent of hGH monomer,
measured using size exclusion HPLC, from hGH liquid formulations of
the present invention after about 53 weeks of storage at 5.degree.
C., as described in Example 11. Formulations 32-37 are formulations
of the present invention and Y & Z are respectively the
simulated NUTROPIN AQ.RTM. and NORDITROPIN SIMPLEXX.RTM.
formulations.
FIG. 11 is a bar graph of the total percent of hGH, measured using
reverse phase HPLC, from hGH liquid formulations of the present
invention after about 53 weeks of storage at 5.degree. C., as
described in Example 11. Formulations 32-37 are formulations of the
present invention and Y & Z are respectively the simulated
NUTROPIN AQ.RTM. and NORDITROPIN SIMPLEXX.RTM. formulations.
FIG. 12 is a bar graph of the total percent of deamidated hGH,
measured using anion exchange HPLC, from hGH liquid formulations of
the present invention after about 53 weeks of storage at 5.degree.
C., as described in Example 11. Formulations 32-37 are formulations
of the present invention and Y & Z are respectively the
simulated NUTROPIN AQ.RTM. and NORDITROPIN SIMPLEXX.RTM.
formulations.
FIG. 13 is a bar graph of the total percent of hGH, measured using
anion exchange HPLC, from hGH liquid formulations of the present
invention after about 53 weeks of storage at 5.degree. C., as
described in Example 11. Formulations 32-37 are formulations of the
present invention and Y & Z are respectively the simulated
NUTROPIN AQ.RTM. and NORDITROPIN SIMPLEXX.RTM. formulations.
DETAILED DESCRIPTION OF THE INVENTION
The therapeutically effective amount of hGH in any given embodiment
of the formulation of the present invention will depend upon the
volume of the formulation to be delivered to any given subject, as
well as the age and weight of the subject, and the nature of the
illness or disorder being treated. When the formulation is to be
delivered to a human subject, the formulation contains at least
about 0.1 mg/ml to about 20 mg/ml hGH, about 0.5 mg/ml to about 15
mg/ml hGH, or about 1 mg/ml to about 10 mg/ml hGH.
The buffer included in the formulation of the present invention
maintains the pH of the formulation at about pH 5 to about pH 7. In
another embodiment, the buffer maintains the pH of the formulation
at about pH 5.7 to about pH 6.5. In yet another embodiment, the
buffer maintains the pH of the formulation at about pH 6. Any
buffer that is capable of maintaining the pH of the formulation
within any pH range given above is suitable for use in the
formulations of the present invention, provided that it does not
react with other components of the formulation to cause visible
precipitates to form after one or more freeze-thaws or after
shipment agitation, or otherwise cause the growth hormone to be
chemically destabilized. The buffer used in the present formulation
comprise a component selected from the group consisting of citrate,
succinate, malate, edetate, histidine, acetate, adipate, aconitate,
ascorbate, benzoate, carbonate, bicarbonate, maleate, glutamate,
phosphate, and tartarate. Particular buffers include either edetate
or citrate as components. Examples of suitable buffers for use in
the formulations of the present invention include, but are not
limited to, sodium citrate, sodium edetate, sodium succinate, and
histidine hydrochloride. Specific embodiments are sodium edetate
and sodium citrate buffers.
The buffer is present in a concentration sufficient to maintain the
pH of the formulation within the pH range described above. The
concentration of buffer in the formulation is about 1 mM to about
100 mM, alternatively about 2 mM to about 50 mM, or alternatively
about 4 mM to about 20 mM.
The non-ionic surfactant and polymer stabilizer used in the
formulation of the present invention are selected for their
capacity to stabilize hGH without causing hGH or other components
of the formulation to precipitate out of solution after undergoing
at least one freeze-thaw event or after undergoing handling and
physical agitation. The non-ionic surfactant may be a polysorbate,
a poloxamer or pluronic, or another ethylene/polypropylene block
polymer. In one particular embodiment the non-ionic surfactant is a
polysorbate, which may be polysorbate 20 and polysorbate 80.
The polymer stabilizer included in the liquid formulation of the
present invention is selected from the group consisting of
polyethylene glycol and polyethylene glycol derivatives. In an a
exemplary embodiment, the polymer stabilizer is polyethylene glycol
of any molecular weight, within an average molecular weight range
of about 400 to about 100,000 kDa, and specifically a molecular
weight range of about 3000 to about 20,000 kDa. Many commercial
forms of poly(ethylene) glycol (also known as "PEG") are available
in these molecular weight ranges, including PEG 400, PEG 3350, PEG
8000, and PEG 20,000. In the formulations of the present invention,
the addition of poly(ethylene) glycols of various molecular weights
has been found to improve the stability of hGH towards physical
agitation as well as freeze-thaws.
The non-ionic surfactant and polymer stabilizer are each present in
a sufficient amount that the surfactant and stabilizer together
stabilize the hGH formulation to physical agitation as well as
freeze-thaws. In another embodiment the amount of surfactant
present in the formulation is an amount that would stabilize the
hGH formulation to physical agitation, even in the absence of the
polymer stabilizer.
The use of non-ionic surfactants such as Polysorbates and
Poloxamers at a concentration of 0.1% or greater to stabilize hGH
liquid formulations has been previously disclosed (U.S. Pat. Nos.
5,763,394 and 5,981,485 (O'Connor et al.; GENENTECH); EP 0955062 A1
(O'Connor et al.; GENENTECH). The only two types of commercially
available hGH liquid formulations utilize Polysorbate 20 at 0.2%
(NUTROPIN AQ.RTM.), and Poloxamer 188 at 0.3% (NORDITROPIN.RTM.).
Unexpectedly, it has been discovered that the liquid formulations
of the present invention provide excellent hGH physical stability,
even with non-ionic surfactant concentrations well below 0.1%.
The non-ionic surfactant in the stable liquid hGH formulation of
the present invention is present at a concentration of at least
about 0.02% (w/w) to about 10% (w/w), alternatively at a
concentration of about 0.04% (w/w) to about 5% (w/w), or
alternatively at a concentration of about 0.05% (w/w) to about 1%
(w/w).
The polymer stabilizer is present at a concentration of at least
about 0.001%, and is suitably present at a concentration of up to
70%. In formulations where it is desired that the viscosity is kept
to a minimum, for example, to facilitate delivery of the
formulation by injection, a relatively low concentration of polymer
stabilizer is used. Under such conditions, the polymer stabilizer
is present at a concentration of about 0.001% to about 20%,
alternatively about 0.01% to about 10%, or alternatively about
0.05% to about 5%.
In another embodiment, the formulation of the present invention
further comprises a tonicity agent. The tonicity agent may also
acts as a further stabilizing agent in the hGH liquid formulation
of the present invention. Suitable tonicity agents include neutral
salts and carbohydrates, such as sugar alcohols, monosaccharides,
and disaccharides. Suitable carbohydrate tonicity agents include
non-reducing mono-, di-, or polysaccharides, or polyols, or neutral
salts, including mannitol, sorbitol, lactitol, xylitol, sucrose
trehalose, sodium chloride and potassium chloride. The carbohydrate
tonicity agent may be mannitol, sorbitol, sucrose or trehalose, or
sorbitol. Formulations of the present invention prepared with each
of the last four tonicity agents have been found to be stable after
exposure to freeze-thaw events, physical agitation and long term
storage. However, sorbitol was found to have a stabilizing effect
on the hGH against physical agitation in the absence of other
stabilizers. This is surprising, since for a previously disclosed
liquid hGH formulation, the use of mannitol as a tonicity agent and
stabilizer has been demonstrated (e.g., U.S. Pat. No. 5,567,677),
and in yet another case, the use of sodium chloride as a tonicity
agent and stabilizer has been demonstrated (see, for example, U.S.
Pat. No. 5,763,394).
When a tonicity agent is present, it may be present in an amount
sufficient to make the formulation isotonic, and suitable for
parenteral injection into a mammal, such as a human subject, into
dermal, subcutaneous, or intramuscular tissues. Depending upon the
concentrations of the other components in the formulation, sorbitol
is present at a concentration of about 50 mM to about 500 mM,
alternatively about 100 mM to about 400 mM, or alternatively about
200 mM to about 300 mM.
In yet another embodiment, the present formulation further
comprises an amino acid stabilizer. Various amino acid stabilizers
have been reported to stabilize proteins, including hGH in liquid
formulations (See, for example use of glycine in U.S. Pat. No.
5,567,677 (Carstensson et al.; PHARMACIA) and use of histidine,
valine, isoleucine, asparagine, and lysine, in various patents
(Sorensen et al., NOVO NORDISK, supra). The amino acid stabilizer
included in the formulation is one that adds further chemical
stability to the formulation upon storage, without causing any
physical instability after one or more freeze-thaw events, or after
exposure to physical agitation. Cysteine was unexpectedly found to
reduce the chemical stability of the hGH liquid formulations of the
present invention, when present therein. Methionine, on the other
hand has been found to improve the chemical stability of hGH in the
present formulations.
The use of methionine as an antioxidant in protein formulations has
been reported in the literature, since proteins tend to undergo
spontaneous oxidation. However, methionine has not been reported to
stabilize hGH in liquid formulations. In fact, it was observed that
methionine did not improve the chemical stability of hGH, nor its
oxidation profile to any significant extent when the polymer
stabilizer component of the present formulation was absent.
Unexpectedly, in the presence of a polymer stabilizer, such as
polyethylene glycol, the addition of methionine was observed to
have a beneficial effect on stability.
In another embodiment, the formulation further comprises a divalent
cation. The formulation is not limited by the nature of the
divalent cation. Exemplary divalent cations are magnesium, calcium,
and zinc. The divalent cation may be a magnesium containing salt,
such as magnesium chloride, magnesium sulfate, or magnesium
hydroxide. The amount of magnesium containing salt in the
formulation is present at a molar concentration that is less than
molar concentration of the buffer, so as not to greatly reduce the
buffering capacity of the buffer by complexation, but sufficiently
high to improve the chemical stability of the formulation.
In another embodiment, the formulation optionally comprises a
preservative, such as phenol and benzyl alcohol. The amounts of
preservative in the formulation is present at a relatively low
concentration that does not chemically or physically destabilize
the hGH, and yet is present at a sufficient concentration that
provides adequate antimicrobial activity for preservative
action.
A particular embodiment of the present invention is a formulation
comprising about 0.1 mg/ml to about 20 mg/ml of a recombinant form
of human growth hormone in an aqueous solution, about 4 to about 20
mM of an edetate or citrate buffer that maintains the formulation
at a pH of about 6, about 0.05% (w/w) to about 1% (w/w) of a
polysorbate surfactant, and about 0.05% (w/w) to about 5% (w/w) of
a polyethylene glycol polymer, wherein the formulation remains
stable after at least one freeze thaw event. In a specific
embodiment the formulation remains stable after at least three
freeze thaw events. In a specific embodiment the formulation
remains stable after at least six freeze thaw events. This
embodiment of the present formulation optionally includes a
tonicity agent, as described above. This embodiment of the
formulation also optionally includes methionine, as described
above. This embodiment of the formulation also optionally includes
a magnesium reagent, as described above. This embodiment of the
formulation further optionally includes a preservative, as
described above.
Formulations of the present invention remain stable after exposure
to a single, and even multiple freeze-thaw events. Formulations of
the present invention also remain stable after exposure to physical
agitation, such as one would expect to encounter upon shipping
product from one location to another. Stability can be measured by
any one of a number of different ways, including visual inspection
for precipitate formation, analysis of percent protein remaining in
solution after exposure to stress conditions (e.g., by
size-exclusion HPLC for hGH monomer or by protein absorbance
analysis for total hGH), or analysis of the formation of chemical
variants of growth hormone (e.g., by anion exchange or reverse
phase HPLC analysis). In one embodiment of the present invention,
no precipitate visible to the naked eye is formed in the
formulation after at least one freeze thaw event. In a specific
embodiment the formulation remains stable after at least three
freeze thaw events. In a specific embodiment the formulation
remains stable after at least six freeze thaw events. In another
embodiment, at least 90%, of the hGH monomer in the formulation
remains in solution as measured by size exclusion HPLC assay after
at least one freeze thaw event.
Formulations of the present invention also provide at least 90% of
hGH monomer in solution by size exclusion HPLC assay, and further
remain fully bioactive after storage for at least 4 weeks at
25.degree. C., or after storage for at least 52 weeks at about 2 to
8.degree. C. Due to their resistance to freeze-thaw conditions,
formulations of the present invention can suitably be stored for
extended periods of time at temperatures below freezing.
Formulations of the present invention also provide at least 90%,
specifically at least 95%, specifically at least 99%, specifically
at least 99.88%, and specifically at least 99.92% recovery of hGH
in solution as measured by size exclusion HPLC.
Formulations of the present invention also provide at least 85%,
specifically at least 86%, and specifically at least 88% recovery
of hGH in solution as measured by reverse phase HPLC.
Formulations of the present invention also provide less than 7%,
specifically less than 6% deamidation in solution as measured by
anion exchange HPLC.
The complete content of all publications, patents, and patent
applications cited in this disclosure are herein incorporated by
reference as if each individual publication, patent, or patent
application were specifically and individually indicated to be
incorporated by reference.
The present invention is further illustrated by the following
examples. These examples are intended to be illustrative of the
invention and should not be used to limit or restrict its
scope.
EXAMPLES
The following examples illustrate one or more of the embodiments of
the formulations of the hGH formulation of the present invention,
described above. In each of the formulations of the present
invention tested below, was somatotropin, a recombinant form of
hGH. The somatotropin used in the Examples below, is the same hGH
protein found in commercial forms of Genotropin.RTM. (PHARMACIA
& UPJOHN COMPANY). For more information about Genotropin.RTM.,
see Physician's Desk Reference, 57.sup.th ed., pub. by Thompson P D
R at Montvale, N.J. (2003). The examples, below, also compare the
physical stability of hGH formulations of the present invention to
the physical stability of known hGH liquid formulations.
Example 1
Physical Stability of Known hGH Liquid Formulations
Somatotropin was used to prepare an aqueous hGH formulation
disclosed in U.S. Pat. No. 5,567,677 (Castensson et al., assigned
to PHARMACIA AB). The hGH formulation had the following
composition: 5 mg/ml hGH, 5 mM sodium citrate, pH 6.2, 12 mM
glycine, and 250 mM mannitol. This formulation is hereinafter
referred to as the "CGM" formulation.
Vials of a commercially available hGH aqueous formulation, NUTROPIN
AQ.RTM. were obtained. The composition of NUTROPIN AQ.RTM.,
according to the product label, was: 5 mg/ml hGH, 10 mM sodium
citrate, 8.7 mg/ml sodium chloride, 2 mg/ml (0.2%) Polysorbate 20,
and 2.5 mg/ml (0.25%) phenol. The composition of this formulation
has also been disclosed in U.S. Pat. No. 5,763,394.
Cartridges of a commercially available hGH aqueous formulation,
NORDITROPIN.RTM. were also obtained. The composition of
NORDITROPIN.RTM., according to the product label, was: 3.3 mg/ml
hGH, 0.67 mg/ml histidine, 40 mg/ml mannitol, 3 mg/ml (0.3%)
Poloxamer 188, and 3 mg/ml (0.3%) phenol.
The three formulations described above were tested for stability
after being exposed to physical stress, including freeze-thaw
events and physical agitation. Freezing was conducted in a
-20.degree. C. freezer; subsequent thawing was conducted at approx
5.degree. C. in a refrigerator; and the process was repeated up to
6 times. The physical agitation test was conducted at approx
5.degree. C. using a mechanical shaker platform at 250 revolutions
per minute ("RPM") for about 20 hours. The physical agitation test
was designed to simulate harsh agitation conditions that may
sometimes occur during shipping. Stability of the formulations was
evaluated by hGH monomer concentration assay using size exclusion
high pressure liquid chromatography (hereinafter, "SE-HPLC") after
each such test. Assay values of greater than 90% were considered
acceptable.
The CGM formulation was found to be stable after exposure to
multiple freeze-thaw events, and essentially 100% of the protein
was recovered by hGH monomer concentration analysis. However, this
formulation was found unstable upon exposure to physical agitation
at 5.degree. C. A cloudy precipitate was formed in the vials after
agitation, and less than 10% of the protein was recovered in
solution by SE-HPLC analysis.
In contrast, the commercially available formulations were found to
be stable after physical agitation but unstable after exposure to
multiple freeze-thaw events. Specifically, both commercial
formulations tested remained clear, and essentially 100% protein
was recovered after forced agitation at 5.degree. C. However, both
of the commercial formulations turned cloudy after only a single
freeze-thaw event, and only about 30% protein was found in solution
by SE-HPLC concentration analysis after exposure to six freeze-thaw
events.
A plot of the results of SE-HPLC analysis of the three formulations
after exposure to either physical agitation or six freeze-thaw
events can be found in FIG. 1. These results are summarized in
Table I, below. As one can see from Table I and FIG. 1, each of the
known hGH liquid formulations tested in this Example is unstable
under conditions of at least one form type of physical stress,
whether that stress is due to physical agitation, or due to
exposure to freezing and thawing.
TABLE-US-00001 TABLE I Stability to Forced Stability to Formulation
Agitation Freeze-Thaws "CGM" No Yes NUTROPIN AQ .RTM. Yes No
NORDITROPIN .RTM. Yes No
Example 2
Physical Stability of hGH Liquid Formulations with and without
Phenol and with a Polysorbate Surfactant and Poly(ethylene)glycol
("PEG")
Four hGH liquid formulations were prepared as shown in Table II,
below, with somatotropin, edetate buffer, polysorbate surfactant,
PEG, and additional excipients. Three concentrations of hGH (1, 5,
and 10 mg/ml) are represented within the first three formulations
(Formulations 1, 2, and 3, respectively), and the fourth
formulation (Formulation 4) contains 5 mg/ml of hGH and 0.3%
phenol, as a preservative.
TABLE-US-00002 TABLE II Formulation Composition 1 2 3 4 hGH 1 mg/ml
5 mg/ml 10 mg/ml 5 mg/ml Concentration Buffer 10 mM 10 mM 10 mM 10
mM Sodium Sodium Sodium Sodium Edetate Edetate Edetate Edetate
Surfactant 0.06% 0.06% 0.06% 0.06% Stabilizer Polysorbate
Polysorbate Polysorbate Polysorbate 20 20 20 20 Polymer 1% PEG 1%
PEG 1% PEG 1% PEG Stabilizer 3350 3350 3350 3350 Preservative -- --
-- 0.3% Phenol Additional 250 mM 250 mM 250 mM 250 mM Excipients
Sorbitol, Sorbitol, Sorbitol, Sorbitol, 10 mM 10 mM 10 mM 10 mM
Methionine, Methionine, Methionine, Methionine, 3 mM 3 mM 3 mM 3 mM
Magnesium Magnesium Magnesium Magnesium Chloride Chloride Chloride
Chloride
As in Example 1, the formulations prepared as described immediately
above were tested for physical stability by SE-HPLC, after exposure
to six freeze-thaws. The formulations were also tested for physical
stability after agitation by shipping the formulations three times
between two cities (Skokie, Ill. and Chesterfield, Mo.), while
keeping them refrigerated at 2.degree. C. to 8.degree. C. using gel
cold packs.
Results of SE-HPLC analysis of each formulation after each physical
stability test described above are illustrated in FIG. 2. Better
than 90% hGH recovery was obtained for all four formulations
tested, demonstrating very good physical stability to freeze-thaws
as well as to agitation. Good stability to freeze-thaws was also
obtained even when phenol was present, in contrast to the results
observed with the commercial phenol-containing hGH liquid
formulations tested as described in Example 1. This was surprising,
considering the fact that phenol is known to promote aggregation of
recombinant hGH (see Maa, Yuh-Fun, et al., supra).
Also surprisingly, good stability to agitation was obtained even
when the surfactant concentration was at 0.06%. This concentration
of surfactant in the hGH formulations tested in this example is
much less than the 0.2 to 0.3% concentration of non-ionic
surfactants in the two commercial hGH liquid formulations tested in
Example 1. It is also considerably less than the concentration
range of 0.1 to 1% claimed in U.S. Pat. No. 5,763,394.
Example 3
Physical and Chemical Stability of hGH Formulations Prepared with
Various Polysorbate Surfactants and PEG of Differing Molecular
Weights
Four liquid formulations of hGH (Formulations 5 through 8) were
prepared as described in Table III, below, formulations containing
5 mg/ml somatotropin, citrate buffer, a polysorbate surfactant, a
PEG polymer, and additional excipients. Three different molecular
weights of PEG (3350, 8000, and 20000) as well as two PEG
concentrations (0.25% and 1%) were included in one of each of the
formulations. A simulated version of the commercially available
NUTROPIN AQ.RTM. was also prepared, according to the formula
provided on the product label, as described in Example 1, above, as
a comparator.
TABLE-US-00003 TABLE III Formulation Composition 5 6 7 8 hGH 5
mg/ml 5 mg/ml 5 mg/ml 5 mg/ml concentration Buffer 5 mM 5 mM 5 mM 5
mM Sodium Sodium Sodium Sodium Citrate Citrate Citrate Citrate
Surfactant 0.06% 0.06% 0.06% 0.06% Stabilizer Polysorbate
Polysorbate Polysorbate Polysorbate 20 20 20 20 Polymer 1% PEG 1%
PEG 1% PEG 0.25% PEG Stabilizer 3350 8000 20000 20000 Additional
250 mM 250 mM 250 mM 250 mM Excipients Sorbitol, Sorbitol,
Sorbitol, Sorbitol, 10 mM 10 mM 10 mM 10 mM Methionine Methionine
Methionine Methionine
As in Example 1, each of the formulations prepared as described in
the present Example, above, was tested for physical stability by
SE-HPLC, after exposure to six freeze-thaw events and to forced
agitation stress. All the Genotropin.RTM. formulations remained
visually clear and better than 90% hGH recovery, as measured by
SE-HPLC, was obtained for all of Formulations 5 through 8 of Table
III, after being subjected to either type of physical stress. In
contrast, in the simulated version of NUTROPIN AQ.RTM., visual
cloudiness was observed after freeze-thaw events; and, on average,
only approximately 70% hGH monomer recovery was obtained.
Each of the formulations was also analyzed by Anion Exchange HPLC
(AEX-HPLC) to evaluate the formation of hGH protein variants after
6 months refrigerated storage at 2.degree. C. to 8.degree. C., and
after 6 weeks storage at 25.degree. C. Although, at least some of
the hGH variants detected by AEX-HPLC were known to be
therapeutically active, this method provided a relative measure of
hGH chemical stability. Specifically, this assay method allows one
to measure the levels of hGH variants formed over time, including
deamidated species.
The results of the AEX-HPLC assay are illustrated in FIG. 3. The
results depicted therein demonstrate that AEX-HPLC detected similar
levels of hGH variants for all the Genotropin formulations at each
temperature. The levels of variants were lower in Formulation 5
through 8, compared to those found in the simulated NUTROPIN
AQ.RTM. formulation (Formulation X in FIG. 3). These results
indicate excellent chemical stability relative to a known hGH
liquid formulation, even apart from excellent physical stability.
The data also demonstrates that different molecular weights and
concentrations of PEG polymer can be used to produce hGH liquid
formulations with similar chemical and physical stability.
Example 4
Physical and Chemical Stability of hGH Formulations Prepared with
Polysorbate Surfactant, PEG, and Various Buffers
Six hGH liquid formulations (Formulations 9 through 14), containing
5 mg/ml somatotropin, various buffers, a polysorbate surfactant, a
PEG polymer, and additional excipients were prepared, as described
in Table IV, below. Six different buffers at 50 mM strength
(citrate, succinate, malate, edetate, bicarbonate and histidine)
were used to prepare one of each of the formulations, at pH 6.
TABLE-US-00004 TABLE IV Formulation Composition 9 10 11 12 13 14
hGH 5 mg/ml 5 mg/ml 5 mg/ml 5 mg/ml 5 mg/ml 5 mg/ml concentration
Buffer 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM Sodium Sodium Sodium
Sodium Sodium Histidine Citrate Succinate Malate Edetate
Bicarbonate Hydro- chloride Surfactant 0.06% 0.06% 0.06% 0.06%
0.06% 0.06% Stabilizer Polysorbate Polysorbate Polysorbate
Polysorbate Polysorbate Pol- ysorbate 20 20 20 20 20 20 Polymer 1%
PEG 1% PEG 1% PEG 1% PEG 1% PEG 1% PEG Stabilizer 20000 20000 20000
20000 20000 20000 Additional 250 mM 250 mM 250 mM 250 mM 250 mM 250
mM Excipients Sorbitol, Sorbitol, Sorbitol, Sorbitol, Sorbitol,
Sorbitol, 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM Methionine Methionine
Methionine Methionine Methionine Methionine
All six formulations tested were found have very good physical
stability. Specifically, all six formulations were found to be
stable after exposure to agitation and to six freeze-thaw events,
as described in Example 2.
The six formulations were also tested for chemical stability using
AEX-HPLC, after 18 weeks of storage at 5.degree. C. The results of
the chemical stability tests are illustrated in FIG. 4. As is shown
in FIG. 4, small differences in chemical stability were observed.
Formulation 12, with edetate buffer, produced the least number of
hGH variants in the chemical stability test. However, the chemical
stability test results indicated that all the formulations were
comparable, suggesting that a variety of buffers could suitably be
used to produce stable hGH liquid formulations of the type
described in Table IV, above.
Example 5
Physical Stability of hGH Formulations with Various Tonicity
Agents
Four hGH liquid formulations (Formulations 15 through 18) were
prepared, as described in Table V, below, with 5 mg/ml
somatotropin, citrate buffer, and with one of each of four
different tonicity agents (mannitol, sorbitol, sucrose and
trehalose). Note that none of the four formulations tested in this
example contained additional stabilizers, such as a polymer
stabilizer or non-ionic surfactant.
TABLE-US-00005 TABLE V Formulation Composition 15 16 17 18 hGH 5
mg/ml 5 mg/ml 5 mg/ml 5 mg/ml concentration Buffer 5 mM 5 mM 5 mM 5
mM Sodium Sodium Sodium Sodium Citrate Citrate Citrate Citrate
Tonicity 250 mM 250 mM 250 mM 250 mM Agent Mannitol Sorbitol
Sucrose Trehalose
Visual particulates were observed in samples of all four
formulations, after forced agitation on a mechanical shaker at 250
RPM for 24 hours at room temperature (at about 25.degree. C.). FIG.
5 is a plot of the percent hGH recovery observed in each
formulation, by protein analysis, after the forced agitation step.
The percent recovery of hGH from the formulation containing
mannitol (Formula 15) was about 90%, a very good recovery rate for
a formulation without any additional stabilizer. However, the best
hGH recovery after agitation (almost 100%) was obtained with
Formulation 16, a formulation prepared with sorbitol. This last
result suggests that sorbitol has a stabilizing effect on hGH by
itself and is a preferable tonicity agent to use in hGH liquid
formulations.
Similar chemical stability was observed between the set of four hGH
liquid formulations prepared with these four tonicity agents, as
described above.
Example 6
Physical Stability of hGH Formulations with less than 0.1%
Concentration of Surfactant
Five different hGH liquid formulations (Formulations 19 through 23)
were prepared, as described in Table VI, below. Each formulation
contained 5 mg/ml somatotropin, citrate buffer, a tonicity agent
(mannitol), and various concentrations of Polysorbate 20 (0, 0.02,
0.04, 0.06 and 0.08%).
TABLE-US-00006 TABLE VI Formulation Composition 19 20 21 22 23 hGH
5 mg/ml 5 mg/ml 5 mg/ml 5 mg/ml 5 mg/ml concentration Buffer 5 mM 5
mM 5 mM 5 mM 5 mM Sodium Sodium Sodium Sodium Sodium Citrate
Citrate Citrate Citrate Citrate Tonicity 250 mM 250 mM 250 mM 250
mM 250 mM Agent Mannitol Mannitol Mannitol Mannitol Mannitol
Surfactant -- 0.02% 0.04% 0.06% 0.08% Polysorbate Polysorbate
Polysorbate Polysorbate 20 20 20 20
As in Example 5, recovery of hGH was monitored by protein
absorbance analysis after forced agitation at room temperature. The
results of this assay are shown in FIG. 6. As shown in FIG. 6, 100%
protein recovery was obtained from a formulations that contained a
polysorbate concentration as low as 0.04% (Formulation 21), and
improved recovery was observed even in formulations where the
polysorbate concentration was as low as 0.02% (Formulation 20).
Example 7
Chemical Stability of hGH Liquid Formulations Containing Methionine
as an Amino Acid Stabilizer
Four formulations of liquid hGH, Formulations 24 through 27, were
prepared as described in Table VII, below. Each formulation
contained 5 mg/ml Genotropin.RTM. hGH protein, citrate buffer for a
pH of 6, polysorbate surfactant, sorbitol, and PEG; with two of the
formulations further containing methionine and two without
methionine.
TABLE-US-00007 TABLE VII Formulation Composition 24 25 26 27 hGH 5
mg/ml 5 mg/ml 5 mg/ml 5 mg/ml concentration Buffer 5 mM 5 mM 5 mM 5
mM Sodium Sodium Sodium Sodium Citrate Citrate Citrate Citrate
Tonicity 250 mM 250 mM 250 mM 250 mM Agent Sorbitol Sorbitol
Sorbitol Sorbitol Surfactant 0.06% 0.06% 0.06% 0.06% Stabilizer
Polysorbate Polysorbate Polysorbate Polysorbate 20 20 20 20 Polymer
1% PEG 1% PEG 1% PEG 1% PEG stabilizer 3350 20000 3350 20000 Amino
Acid -- -- 10 mM 10 mM Stabilizer Methionine Methionine
Chemical stability of each of the four formulations described
immediately above was evaluated by reverse-phase HPLC (RP-HPLC).
Similar to AEX-HPLC, RP-HPLC allows detection of hGH protein
variants. Although these variants are known to be therapeutically
active, the method provides a relative measure of hGH chemical
stability.
FIG. 7 is a plot of the results of the chemical stability analysis.
As shown in FIG. 7, after 31 weeks at 5.degree. C., RP-HPLC
analysis indicated that the hGH formulations 26 and 27 that
contained methionine had lower levels of protein variants as
compared to formulations 24 and 25 that did not have methionine.
The presence of added methionine did not have any stabilizing
effect on hGH liquid formulations prepared earlier without PEG.
Unexpectedly, however, it was found that methionine improved the
chemical stability of the hGH liquid formulations tested in this
Example, in which PEG polymer was not present. It is expected that
formulations of the present invention could be also combined with
other amino acid stabilizers (e.g. histidine, leucine, valine, and
asparagine) to further stabilize hGH liquid formulations.
Example 8
Chemical Stability of hGH Formulations Containing Magnesium as a
Divalent Cation Stabilizer
Four hGH liquid formulations were prepared (Formulations 28 through
31), as described in Table VIII, below. Each formulation contained
5 mg/ml somatotropin, buffer, polysorbate surfactant, polymer
stabilizer, and amino acid stabilizer. Two of the formulations
(Formulations 29 and 31) also contained a Magnesium reagent, while
the other two (Formulations 28 and 30) were prepared without
Magnesium.
TABLE-US-00008 TABLE VIII Formulation Composition 28 29 30 31 hGH 5
mg/ml 5 mg/ml 5 mg/ml 5 mg/ml concentration Buffer 5 mM 5 mM 10 mM
10 mM Sodium Sodium Sodium Sodium Citrate Citrate Citrate Citrate
Surfactant 0.06% 0.06% 0.06% 0.06% Stabilizer Polysorbate
Polysorbate Polysorbate Polysorbate 20 20 20 20 Polymer 1% PEG 1%
PEG 1% PEG 1% PEG stabilizer 20000 20000 3350 3350 Additional 250
mM 250 mM 250 mM 250 mM Excipients Sorbitol, Sorbitol, Sorbitol,
Sorbitol, 10 mM 10 mM 10 mM 10 mM Methionine Methionine Methionine
Methionine Magnesium -- 2.5 mM -- 3 mM Reagent Magnesium Magnesium
hydroxide chloride
Formulations 28 and 29 were analyzed by RP-HPLC, after 9 weeks
storage at 25.degree. C. Formulations 30 and 31 were analyzed after
8 weeks storage at 25.degree. C. In both sets of formulations
tested, lower levels of protein variants were observed in
formulations where magnesium reagent was present (29 and 31),
indicating improved hGH stability in the presence of magnesium.
These results are surprising because prior disclosures have
described the stabilizing effect of calcium and zinc on hGH
formulations (see U.S. Pat. No. 6,022,858), but not of magnesium.
It is expected that formulations of the present invention could
also be suitably combined with other divalent cations, such as
calcium and zinc ions.
Example 9
Bioactivity of hGH Formulations Prepared with Buffer, Non-ionic
Surfactant, Polymer Stabilizer and Additional Excipients
Formulations 5, 6, and 8 from Example 3, above, were tested for
bioactivity after 6 weeks storage at 25.degree. C. by injecting
once daily into separate hyposectomized rats using a bioassay
method for hGH that complies with the European Pharmacopoeia.
Phosphate buffered saline (PBS) was injected as a control. The
results of this study are illustrated in FIG. 9 and confirm that
the formulations retained full bioactivity, which is to be expected
if the formulations have adequate stability upon storage. All the
hGH formulations resulted in expected level of rat growth whereas
the control formulation (PBS) did not cause growth.
Example 10
Antimicrobial Effectiveness of hGH Formulations Containing Phenol
as a Preservative
Formulation 4 from Example 2 (see Table II), containing 5 mg/ml
hGH, 10 mM sodium edetate buffer, 0.06% polysorbate 20, 1% PEG
3350, 0.3% phenol, and additional excipients, was tested for
antimicrobial effectiveness against two representative
microorganisms (E. coli and A. niger). The formulation had adequate
antimicrobial activity as per acceptance criteria described in
United States Pharmacopoeia, demonstrating that a preservative can
be optionally added to the formulation with expected antimicrobial
activity.
Example 11
Long-Term Storage of Liquid Genotropin Formulations
Six liquid formulations of hGH (Formulations 32 through 37) were
prepared as per the Table IX, below, formulations contain 5 mg/mL
somatotropin, citrate or edetate buffer (pH 6.0), 250 mM Sorbitol,
(0.06% w/w) polysorbate 20, (1% w/w) PEG 3350 polymer, 10 mM
methionine, and some of the formulations included magnesium
chloride and some contained phenol as a preservative. Simulated
versions of the commercially available NUTROPIN AQ.RTM. and
NORDITROPIN.RTM. were also prepared, according to the formula
provided on the product label (Y & Z). The composition of
NUTROPIN AQ.RTM., according to the product label, was: 5 mg/ml hGH,
10 mM sodium citrate, 8.7 mg/ml sodium chloride, 2 mg/ml (0.2%)
Polysorbate 20, and 2.5 mg/ml (0.25%) phenol. The composition of
this formulation has also been disclosed in U.S. Pat. No.
5,763,394. The composition of NORDITROPIN.RTM., according to the
product label, was: 3.3 mg/ml hGH, 0.67 mg/ml histidine, 40 mg/ml
mannitol, 3 mg/ml (0.3%) Poloxamer 188, and 3 mg/ml (0.3%)
phenol.
TABLE-US-00009 TABLE IX Sodium Disodium Citrate Edetate MgCl.sub.2
Phenol Formulation # (mM) (mM) (mM) (% w/w) 32 10 -- -- -- 33 -- 10
-- -- 34 10 -- 3 -- 35 -- 10 3 -- 36 10 -- 3 0.3 37 -- 10 3 0.3
Each of the formulation prepared as described in the present
Example, above, was kept at proposed storage condition (2-8.degree.
C.) for 53 weeks. Samples were analyzed at 8, 16, 28 and 53 weeks.
At each time point, samples were analyzed visually for presence of
particulates, change in color, and clarity. pH measurements were
also conducted. Presence of aggregates was monitored by SE-HPLC.
All formulations, tested in this Example, remained visually clear,
colorless and free of particles and did not show any significant
change in pH. In addition, better than 99% hGH recovery, as
measured by SE-HPLC, was obtained for all Formulations 32 through
37 of Table IX, and all the comparators tested in this Example,
after being subjected to storage at 2 to 8.degree. C. for 53 weeks
(FIG. 10).
Each of the formulations was analyzed by Reverse Phase HPLC
(RP-HPLC) to evaluate the formation of hGH protein variants after
53 weeks of refrigerated storage (5.degree. C.). Although, at least
some of the hGH variants detected by RP-HPLC are known to be
therapeutically active, this method provides a measure of hGH
recovery and can be used as an indication of hGH % purity.
The results of the RP-HPLC assay are illustrated in FIG. 11. The
results depicted therein clearly demonstrate that all the
Genotropin formulations reported higher hGH % recovery after
storage at 2 to 8.degree. C. for 53 weeks. Formulations 33 & 35
(edetate buffer) reported the highest recovery % followed by
Formulations 32 & 34 (citrate buffer). Genotropin formulations
containing preservative (0.3% phenol) showed recovery in the range
of 86.9-87.3%. In contrast, the simulated comparator products
(NUTROPIN AQ.RTM. and NORDITROPIN.RTM.) showed recovery in the
range of 85.5-87.3%.
Each of the formulations was also analyzed by Anion Exchange HPLC
(AEX-HPLC) to evaluate the formation of hGH protein variants after
53 weeks of refrigerated storage (5.degree. C.). This method
provides a good measure of hGH chemical stability; specifically
this assay method allows one to measure the levels of hGH variants
formed over time, including deamidated species.
The results of the AEX-HPLC assay are illustrated in FIG. 12 (total
deamidation) and FIG. 13 (hGH recovery). The results depicted
therein demonstrate that AEX-HPLC detected lesser or similar levels
of hGH variants for all Genotropin formulations than the
comparators. The levels of variants are lower in Formulations 32
through 35 and 37, compared to those found in the simulated
NUTROPIN AQ.RTM..RTM. and NORDITROPIN.RTM..RTM. (Formulations Y
& Z in FIGS. 12 & 13). These results indicate that the
Genotropin formulations show excellent chemical stability and
physical stability relative to the two known, commercially
available, hGH liquid formulations.
* * * * *